Abstract Peripheral neuropathic pain is a major health problem in the U.S. because of its high prevalence and poor response to currently available therapies. Although the pain has many pathological causes and anatomical locations, and its presentation in patients can vary considerably, fundamentally, its positive symptoms include just two distinct features: spontaneous pain and stimulus-evoked pain. Spontaneous pain arises as a consequence of ectopic activity in axons in the injured nerve?action potentials arising spontaneously from hyperexcitable membranes. Surprisingly, it is not at all clear what the exact source of this activity is?whether it originates in injured neurons or in neighboring intact ones, in A- or C-fibers, and in nociceptors or non- nociceptors. Furthermore, although there are changes in ion channel expression after peripheral axonal injury, the molecular mechanisms responsible for ectopic activity are still uncertain. It is also not known whether there are different mechanisms in different sensory neuron subtypes and whether these change over time. The goal of this grant is to precisely elucidate the cellular source and molecular mechanisms of spontaneous somatic pain after sciatic nerve injury in mice using live imaging in vivo, single cell Next Generation profiling by Nuc-seq and by Patch-seq - single cell profiling of electrophysiologically characterized pre-injured and intact neurons in vitro, and by measuring spontaneous changes in behavior in freely moving animals in an automated observer independent way. Finally, the project will also evaluate whether silencing specific subsets of sensory neurons using genetic tools that block synaptic input into the CNS has an effect on spontaneous pain-related behaviors. These experiments will provide data on the timing, extent and pattern of spontaneous activity after nerve injury, and in which sensory neurons. They will also identify those transcriptional changes specific to the presence of spontaneous activity at a single cell level. This mechanistic understanding will lay a foundation for identifying novel therapeutic opportunities for treating spontaneous neuropathic pain.